Impaired Membrane Transport in Methotrexate-resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier

Abstract

The basis for impaired reduced folate carrier (RFC) activity in methotrexate-resistant CCRF-CEM (CEM/ Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3.1-kilobase RFC transcript. A ϳ85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/ Mtx-1 transcripts at positions 130 (P 1 ; changes glycine 44 3 arginine) and 380 (P 2 ; changes serine 127 3 asparagine Despite the availability of newer antifolates, methotrexate (Mtx) 1 continues to play an important role as an antineoplastic agent. To reach its intracellular target, dihydrofolate reductase, the preferred route of Mtx entry involves the reduced folate carrier (RFC; 1, 2). RFC transport of Mtx is critical to drug action because of its role in generating sufficient unbound intracellular antifolate to sustain maximal enzyme inhibition (1). Furthermore, high levels of Mtx are also necessary for the synthesis of Mtx polyglutamates (1).Defective membrane transport of Mtx by RFC has been identified as a major mechanism of Mtx resistance (1-11). Transport alterations can manifest as reduced rates of carrier translocation (reduced V max ), decreased affinities for transport substrates (increased K t ), or both, and may involve decreased levels of normal RFC (6) or the expression of structurally altered RFC proteins (7-11). For instance, in Mtx-resistant K562 (K500E) cells, impaired Mtx transport is accompanied by decreased RFC transcripts and protein (6). A G to A transition at position 890 of the murine RFC cDNA resulted in a substitution of serine 297 by asparagine and a selective decrease in Mtx binding affinity (ϳ4-fold) without effects on other antifolate analogs (aminopterin, Ref. 9). Likewise, replacement of serine 46 by asparagine (10) or glutamate 45 by lysine (11) in murine RFC resulted in greater impairment of uptake for Mtx than (6S)-5-formyl tetrahydrofolate. In severely transport defective L1210 cells (Mtx r A), loss of transport activity appeared to reflect a single (G to C) point mutation at nucleotide 429 of the murine RFC cDNA sequence which resulted in the substitution of proline 130 by alanine (7). However, these cells also contained a wild-type RFC allele that was not transcribed. A silent wild-type RFC allele was described for Mtx-resistant MOLT-3 cells (MOLT-3/Mtx 10,000 ; Ref. 8). Moreover, two mutations in the RFC coding region were detected which resulted in the creation of new stop codons and synthesis of truncated nonfunctional RFCs (8).In this report, the molecular mechanisms responsible for the transport-impaired phenotype (ϳ3% of wild-type) of Mtx-resistant (ϳ243-fold) CCRF-CEM (CEM/Mtx-1;12) cells were examined. We show that although the levels of RFC transcripts are essentially unchanged from wild-type cells, there is a complete loss of RFC protein due to ear...

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

“…High levels of both wild-type and E45K hRFCs were detected in plasma membranes of transfected CEM/MTX R0 cells by Western blotting, suggesting that there were no significant differences in targeting or stability between the carrier forms. 21,40 Both transporters were functional, with overtly similar properties. There were minor differences in relative affinities for MTX binding (ie K t s) and in absolute rates of carrier translocation (V max ) between the transfected lines, and with parental CCRF-CEM cells, although they could largely be explained by the differences in levels of carrier expression (20-fold for CEM R0/RFC and 40-fold for CEM R0/E45K of parental levels).…”

Resistance to antifolates

“…The first TMD appears to be a favored locus of mutations in acquired resistance to antifolates, further supported by the mutations at this site in a variety of CCRF-CEM cell lines selected for resistance to antifolates in the absence of a mutagen (Jansen et al, 1998;Drori et al, 2000;Rothem et al, 2002). Beyond single point mutations within the open reading frame, a variety of other mutations have been detected in MTX-resistant cell lines including mutation of the ATG start codon, insertions and frameshifts, truncated proteins, deletions, mutations resulting in RFC instability, and loss of RFC alleles due to translocations (Wong et al, 1999;Zhao et al, 1999b;Ding et al, 2001;Rothem et al, 2002).…”

Design, Synthesis, and Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors

“…The RFC is the most important carrier, and impairment of the RFC system in tumor cells also leads to drug resistance. [21][22][23][24][25] In general, the problem of resistance in tumors, due to low or defective FPGS, has placed limitations on the use of classical antifolates, which depend on polyglutamylation for their antitumor effects. To overcome these problems associated with classical antifolates, lipophilic nonclassical antifolates were also designed and synthesized.…”